2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This unit addresses the developmental origins of obesity, cardiovascular disease, and other chronic diseases of nutritional lineage and contains four individual research projects:. 1)The role of cholesterol in regulation of the Hedgehog developmental pathway;. 2)the role of GATA protein complexes in adipocyte;. 3)nutritional influences on epigenetic gene regulation during development; and. 4)nutritional influences on mammalian developmental epigenetics. These projects directly addresses the aims of NP 107 (Human Nutrition) Component 2 (Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease) Priority Objective C (Identify the nutrient-relevant genetic, epigenetic, and environmental influences on gene expression and developmental programming that have permanent consequences on human health and disease. Identify the critical periods of development in which these effects are manifested). Additionally, these projects fit into the ARS Strategic Plan Goal 4, Improve the Nation's Health, specifically Objective 4.1: Promote Healthier Individual Food Choices and Lifestyles and Prevent Obesity; Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods; Determine Food Consumption Patterns of Americans. Project 1: The role of cholesterol in regulation of the Hedgehog developmental pathway Hedgehog (Hh) signaling pathway is fundamental for early embryonic patterning of many structures, including the neural tube, axial skeleton, limbs, lungs, and gut. There is growing evidence that germline mutations in various genes in the Hh signaling pathway cause developmental defects, and inappropriate activation of the pathway can result in cancer in susceptible tissues. Recent data have linked this pathway to several gut malformations, including malrotation, imperforate anus, and a Hirshsprung-like phenotype in mice. This project focuses on the critical role of dietary cholesterol in early embryonic patterning of the fetus and continued control of cell growth and differentiation later in life. In contrast to the national focus on cholesterol as an element predisposing to cardiovascular disease (Nutrition, Cardiovascular Health And Genomics), this research will pursue cholesterol as a key nutritional element required for coordinated cell signaling and appropriate human embryonic development. Characterization of the Ptc promoter, especially with regard to regulation by sterol or retinoid elements, will help identify both the dietary and genetic influences on gene expression and developmental programming. Understanding how alternative splicing of the Ptc gene may be involved in regulating tissue-specific expression will identify mechanisms responsible for embryonic development and possible malformations. A coordinated approach using both in vitro expression systems and animal models will be used to identify the interactions linking nutrition and genetics in the regulation of this pathway. This information may lead to potential targets for the prevention of birth defects and cancer. It may also suggest a role for nutritional imprinting in the developing fetus, underscoring the importance of appropriate maternal dietary intake during gestation and aggressive nutritional support of the premature infant. The most important product of these studies will be fundamentally new information about the role of cholesterol as a genetic and developmental regulator. This information is likely to be used to help develop therapeutic targets for certain developmental syndromes and cancers linked to the Hh signaling pathway. The primary consumers of the genetic and scientific information generated by these studies will be healthcare professionals (such as neonatologists, obstetricians, genetic counselors, and oncologists), scientists, and pharmaceutical-based drug developers. Project 2: The role of GATA protein complexes in adipocyte The study of adipogenesis may provide potential prevention and treatment methods of obesity and associated metabolic diseases. During previous research, it was demonstrated that both GATA-2 and GATA-3 transcription factors are expressed in preadipocytes and constitutive expression of either GATA-2 or GATA-3 suppresses adipocyte differentiation. It is also known that GATA factors interact with many proteins, some of which (Rb, C/EBP, and Trap220) are critical for adipogenesis. The characterization of GATA protein complexes may provide new insights into the mechanism of adipogenesis and obesity. We will determine the protein levels and subcellular distribution of GATA-2, GATA-3 and their associated proteins in the adipocyte differentiation process. In this project, we will purify the GATA protein complexes and identify the constituents and dynamics of the GATA protein complexes during adipogenesis and characterize their effects on adipocyte differentiation. The study of adipose tissue development could provide important clues to understand the molecular mechanisms of obesity and related metabolic diseases. Project 3: Nutritional influences on epigenetic gene regulation during development Nutritional deficiency during fetal life may impose a "metabolic memory" on the body that can result in predisposition to chronic adult-onset disorders such as obesity, cardiovascular disease, adult metabolic syndrome and neuropsychiatric disorders, which combined affect a large fraction of the population. An important contributing molecular mechanism responsible for this "metabolic memory" may be methylation of cytosine (one of the bases of DNA), when it is followed by guanine (CG). We propose that the supply of vitamins and nutrients such as folic acid and betaine ("methyl donors") can influence DNA methylation levels, because they function in the metabolic pathway that generates the universal methyl donor s-adenosyl methionine (SAM) used in DNA methylation. The identification of the epigenetic effects of dietary methyl donor supplementation could lead to further targeted studies in humans. This may result in changed recommendations for nutrition and specific supplements in pregnancy or early childhood. The food industry, the general public, women of reproductive age and during pregnancy, and other researchers will all benefit from the knowledge gained in this project. Project 4: Nutritional influences on mammalian developmental epigenetics It is likely that nutrition during prenatal and early postnatal development has permanent effects on epigenetic gene regulation in humans. Our understanding of these phenomena, however, is rudimentary. Mechanistic studies in animal models are urgently needed to enable the formulation of specific hypotheses that can be tested in humans. The overall hypothesis of the proposed research is that maternal dietary methyl donor supplementation before conception and during pregnancy alters DNA methylation of specific genomic regions in the early embryo, and that these alterations persist to adulthood. Because epigenetic dysregulation is implicated in a broad range of human disease, such induced epigenetic alterations may enable early nutrition to influence adult metabolism and chronic disease susceptibility. Scientists interested in understanding nutritional influences on mammalian epigenetics will benefit directly from this research, the results of which will enable the formulation of specific hypotheses that can be tested in human populations. Human populations in the US and worldwide may ultimately benefit from the identification of short-term dietary interventions that have a long-term impact on health. 2.List the milestones (indicators of progress) from your Project Plan. Project 1: The role of cholesterol in regulation of the Hedgehog developmental pathway Year 1 (2005) Perform baseline trafficking studies using Bioptechs perfusion chamber system, cav-1 muts, cholesterol staining of membranes; Shh binding studies Complete mutational analysis of GliREs, characterization of two SP-1 sites, and c/EBP site using primer deletion, luciferase reporter assays, gels shift and Supershift experiments Complete characterization of mouse 1B and 1 promoter regions, generate probes for in situ hybridization studies Year 2 (2006) Initiate fractionation and co-immunoprecipitation studies on cav null and cholesterol depleted cells to show biochemical interactions between Hh receptor components and cav-1 mutants Initiate identification and characterization of RA response elements, complete characterization of human exon 1 and Exon 1A promoter regions, begin rexinoid partner studies, begin SRE/SREBP studies Initiate whole mount and tissue in situ hybridization for Exon 1B and 1 transcripts, begin quantitative RT-PCR and Northern analysis of RNA splice form expression Year 3 (2007) Complete fractionation and co-immunoprecipitation studies on cav null and cholesterol depleted cells to show biochemical interactions between Hh receptor components and cav-1 mutants Complete identification and characterization of RA response elements, complete characterization of human exon 1 and Exon 1A promoter regions, begin rexinoid partner studies, begin SRE/SREBP studies Complete whole mount and tissue in situ hybridization for Exon 1B and 1 transcripts, begin quantitative RT-PCR and Northern analysis of RNA splice form expression Year 4 (2008) Initiate functional assays using luciferase Gli reporter in cav-1 null and cholesterol depleted cells Initiate RA/rexinoid studies, complete SRE/SREBP identification and characterization Initiate in situ hybridization, QRT-PCR and Northern analysis of RNA splice form expression sequentially during development and in tumorigenesis Year 5 (2009) Complete functional assays using luciferase Gli reporter in cav-1 null and cholesterol depleted cells Complete RA/rexinoid studies, complete SRE/SREBP identification and characterization Complete in situ hybridization, QRT-PCR and Northern analysis of RNA splice form expression sequentially during development and in tumorigenesis Project 2: The role of GATA protein complexes in adipocyte Year 1 (2005) Determine protein level changes and subcellular distribution of GATA-2 and GATA-3 in cultured preadipocyte and differentiated adipocytes and murine preadipocytes and adipocytes. Construct TAP-GATA-2 plasmid. Establish TAP-GATA-2 expressing 3T3-F442A and 3T3-L1 cells. Year 2 (2006) Identify the constituents of the GATA-2 complexes by mass spectrometry. Perform TAP protein complex purification and identification of individual components of the protein complexes purified Year 3 (2007) Perform gel filtration purification of GATA protein complexes and cross confirm the results with the TAP method. Perform GST pull down assay to confirm the protein-protein interaction between individual GATA protein complex constituents. Determine the architecture of the GATA-2 protein complexes by mapping the regions responsible for the interaction between GATA-2 and its associated proteins. Year 4 (2008) Produce deletion or single amino acid residue mutations of known or newly identified GATA associated proteins and perform GST pull down assay on those mutants. Determine protein level changes and subcellular distribution of newly identified GATA-2 associated proteins in cultured preadipocyte and differentiated adipocytes and murine preadipocytes and adipocytes. Characterize the roles that GATA protein complexes play in adipose tissue development and obesity. Year 5 (2009) Initiate the generation of transgenic and/or knockout mice. The full characterization of the phenotypes and underlying mechanism may proceed to the following years Determine the effects of GATA protein complex components on adipogenesis by ectopic expression and RNA1. Project 3: Nutritional influences on epigenetic gene regulation during development Year 1 (2005) Perform diet pilot studies using six methyl-donor-supplemented (HM) or depleted (LM) diets and perform analysis of relevant metabolites. Year 2 (2006) Perform metabolic, pathological and biochemical evaluations at various stages after birth on mice that were exposed during development to the selected best experimental LM or HM diet to find metabolic alterations that indicate altered methylation. Year 3 (2007) Perform methylation-sensitive restriction landmark genome scanning (RLGS) on tissues (primarily brain) from these mice and analyze data to find which CpG islands and nearby genes have LM or HM diet-induced altered methylation Year 4 (2008) Perform RLGS spot confirmation analysis to confirm at specific individual CpG sites the altered methylation that was found on the screen. Initiate microarray-based gene expression screening on tissues from mice exposed to the best LM and HM diets in utero to find genes with altered expression. Year 5 (2009) Perform methylation analysis of repetitive elements in the genome to find endogenous repeats and transposons with altered methylation. Analyze and confirm results from gene expression microarray experiments and integrate the expression data with methylation data. This is expected to yield a number of methylation-variable loci that can be further studied in detail. Project 4: Nutritional influences on mammalian developmental epigenetics Year 2 (2006) Conduct diet studies with AxinFu mice; collect tissues Perform genotyping and methylation studies for AxinFu study Validate HPLC techniques for measurement of SAM, SAH and HCY Year 3 (2007) Conduct bioinformatics analysis to identify IAP-promoted genes Assess interindividual variation in methylation status at these loci Perform bioinformatics analysis examining correlates of interindividual variation Conduct methyl supplementation studies with C57 x Cast matings; collect tissues Perform genomic sequencing to identify C57/Cast polymorphisms Conduct Phase 1 supplementation studies; collect tissues Assess tissues of a/a females for Phase 1 diet effects on SAM, SAH, and HCY Optimize embryo isolation techniques Optimize bisulfite sequencing approaches for low-copy number DNA Assess Avy CpG methylation in offspring showing coat color effects Initiate collection of oocyte, sperm, and embryo tissues Year 4 (2008) Measure allelic expression of imprinted genes in tissues from offspring Measure allelic methylation at imprinted genes with allelic expression changes Conduct Phase 2 supplementation studies; collect tissues Assess tissues of a/a females for Phase 2 diet effects on SAM, SAH, and HCY Complete collection of oocyte, sperm and embryo tissues Year 5 (2009) Measure Avy CpG methylation in offspring showing coat color effects Measure Avy CpG methylation in collected tissues 4a.What was the single most significant accomplishment this past year? Project 3: Nutritional influences on epigenetic gene regulation during development ADVANCEMENTS THAT LEAD TO GLUCOSE INTOLERANCE Researchers at the Children's Nutrition Research Center in Houston, TX, have observed that our Mecp2R308/Y mutant mice when fed with a "low methyl donor" or "high methyl donor” diet appear to develop glucose intolerance as they age when compared with Mecp2R308/Y mutant mice on regular diet, or to wild-type mice on all three diets. This supports that this model is sensitive to dietary levels of methyl donors, resulting in a metabolic change that may predispose to diabetes. These mice were exposed throughout life, starting in utero, to these diets. Additional investigation will determine if "in utero only" exposure also leads to glucose intolerance in this model. 4b.List other significant accomplishments, if any. Project 1: The role of cholesterol in regulation of the Hedgehog developmental pathway UNDERSTANDING EMBRYONIC DEVELOPMENT OF BRAIN, LIMBS, AND VASCULAR STRUCTURES Further investigation of the embryonic development of the brain, limbs, and vascular structures is needed. Scientists at the CNRC in Houston, TX, successfully identified three alternate splice forms of the Hedgehog receptor Ptc in the early mouse embryo by both RT-PCR and in situ hybridization. In general, the expression of these individual splice forms in embryonic tissues were non-overlapping, indicating that they each have unique contributions/importance in early embryonic development. During these early mouse embryonic time periods, expression of specific Exon 1B and Exon 1C splice forms were concentrated in the developing brain, neural tube, limbs and lungs, while Exon 1 was expressed at lower levels and appeared concentrated in the heart/vascular areas. These studies are important because they will add to the understanding of normal and aberrant embryonic development of the brain, limbs, and vascular structures. Many disorders seen in human fetuses/neonates have components of early embryonic maldevelopment which contribute to infant mortality and long-term disability. MODULATING THE HEDGEHOG SIGNAL FOR EMBRYONIC DEVELOPMENT Further investigation of the embryonic development of the brain, limbs, and vascular structures is needed. In addition to the identification of two novel mice Ptc splice forms, Exon 1C and Exon 1A, researchers at the Children's Nutrition Research Center, Houston, TX, have also characterized a short form of Ptc Exon 1C that produces a condensed Ptc protein. This protein may be used by the cell to help modulate the Hedgehog signal, thereby reducing activity of the pathway as a whole. Pathway control is particularly important because unregulated pathway activity (increased or decreased) has been shown to cause cancer in susceptible tissues and embryonic malformations. Project 2: The role of GATA protein complexes in adipocyte STIMULATING MITOCHONDRIAL GENE EXPRESSION WITHOUT CALORIC RESTRICTION In our pursuit to identify the molecular mechanism of the life extending effects of caloric restriction, scientists at the Children's Nutrition Research Center in Houston, TX, have discovered the SIRT3 gene, as a regulator of adaptive thermogenesis in response to caloric restriction and cold exposure. The SIRT3 gene stimulates mitochondrial gene expression and respiration, and reduces cellular oxidative stress. Due to the findings that the enzymatic activities of SIRT3 are essential for its action, it might be possible to develop specific activators to boost the activity of SIRT3 and achieve its beneficial effects without caloric restriction. Therefore, if SIRT3 plays a role in delaying aging and reduces diabetes risk, researchers might be able to develop a drug to activate its function. GATA PROTEIN EXPRESSED HIGHLY IN WHITE ADIPOSE In our study of the mechanism control process of adipocyte formation, we found that the PU.1 transcription factor, a known GATA interacting protein, is expressed highly in the white fat tissue, as well as in the lung, but quite low in the brown fat tissue. In white fat tissue, PU.1 is preferably expressed in the stromal-vascular fraction, which contains preadipocytes, but not the adipocyte fraction. Furthermore, the expression of PU.1 is increased in the white adipose of several genetic obese mice models. 4c.List any significant activities that support special target populations. None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. These projects directly addresses the aims of NP 107 (Human Nutrition) Component 2 (Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease) Priority Objective C (Identify the nutrient-relevant genetic, epigenetic, and environmental influences on gene expression and developmental programming that have permanent consequences on human health and disease. Identify the critical periods of development in which these effects are manifested). Additionally, these projects fit into the ARS Strategic Plan Goal 4, Improve the Nation's Health, specifically Objective 4.1: Promote Healthier Individual Food Choices and Lifestyles and Prevent Obesity; Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods; Determine Food Consumption Patterns of Americans. Project 1: The role of cholesterol in regulation of the Hedgehog developmental pathway CNRC Researchers have identified two novel splice forms of the mouse Hedgehog receptor Ptc, named Exon 1C and Exon 1A. CNRC Researchers have also characterized critical regions of the Exon-specific promoter regions, which helps identify targets for regulation and tissue specificity. We have generated specific RNA probes for each of the splice forms so that their expression and tissue specificity during embryonic development can be mapped. This information is critical to understanding the unique contributions of each splice form to organ system/embryonic development. Because these genes/splice forms are incredibly conserved (greater than 97%) between mouse and human, these studies will provide information applicable to human embryonic development and disease. Project 2: The role of GATA protein complexes in adipocyte In our study of the mechanism control the process of adipocyte formation, we found that the PU.1 transcription factor, a known GATA interacting protein, is expressed highly in the white adipose, as well as in the lung, but quite low in the brown adipose tissue. In white adipose tissue, PU.1 is preferably expressed in the stromal-vascular fraction, which contains preadipocytes, but not the adipocyte fraction. Furthermore, the expression of PU.1 is increased in the white adipose of several genetic obese mice models. In our pursuit to identify the molecular mechanism of the life extending effects of caloric restriction, Scientists at the Children's Nutrition Research Center in Houston, TX, have discovered the SIRT3 gene as a regulator of adaptive thermogenesis, in response to caloric restriction and cold exposure. The SIRT3 gene stimulates mitochondrial gene expression, respiration, and reduces cellular oxidative stress. Additionally, researchers have found that the action of SIRT3 is mediated by PGC-1. Due to the findings that the enzymatic activities of SIRT3 are essential for its action, it might be possible to develop specific activators to boost the activity of SIRT3 and achieve its beneficial effects without caloric restriction. Therefore, if SIRT3 plays a role in delaying aging and reduces diabetes risk, researchers might be able to develop a drug to activate its function. Project 3: Nutritional influences on epigenetic gene regulation during development We observed that Mecp2R308/Y mutant mice fed diets with varying methyl donor content (HM and LM) develop glucose intolerance. This suggests that methyl-donor levels may indeed predispose to diabetes. An additional accomplishment is progress on the development of a microarray for genome-wide screening for DNA methylation levels. This is predicted to become an important tool for future goals of this project. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? None. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Project 1: The role of cholesterol in regulation of the Hedgehog developmental pathway Harris, L.L., Bukowski, J.T., Karpen, H.E. 2005. Differential expression of alternate Hedgehog receptor splice forms during development. PAS-SPR 2005, May 2005, Washington, D.C. (poster). Project 4: Nutritional influences on mammalian developmental epigenetics Waterland, R. 2004. Does nutrition during infancy and early childhood contribute to later obesity via metabolic imprinting of epigenetic gene regulatory mechanisms? The 56th Nestle Nutrition Workshop Feeding During Late Infancy and Early Childhood: Impact on Health, November 2004, Noordwijk, The Netherlands. Waterland, R. 2005. Do early nutritional influences on developmental epigenetics contribute to adult-onset chronic disease susceptibility? The Mars Nutrition Research Council meeting The Metabolic Syndrome in Children, January 2005, Houston, Texas. Waterland, R. 2005. Nutritional influences on developmental epigenetics. The CNRC Current Topics in Neonatal/Infant Nutrition and Metabolism symposium Methionine, Methylation and Epigenetics, January 2005, Houston, Texas. Waterland, R. 2005. Early nutritional influences on developmental epigenetics. The Child Health Foundation meeting The Future of Infant Nutrition, January 2005, Obergurgl, Austria. Waterland, R. 2005. Do early nutritional influences on developmental epigenetics affect adult chronic disease susceptibility? The International Life Sciences Institute annual meeting, January 2005, New Orleans, Louisiana. Waterland, R. 2005. Early nutrition, epigenetics, and adult disease. The International Union of Physiological Sciences satellite symposium, The Prenatal Environment, Programming and Postnatal Consequences, March 2005, San Diego, California. Waterland, R. 1005. Epigenetic mechanisms and postnatal GI development. Mead Johnson Nutritionals 25th Anniversary Symposium of the Freedom to Discover Nutrition Program Nutrition and Development of the Gastro-Intestinal Tract, June 2005, Cincinnati, Ohio. Waterland, R. 2005. Memories of when we were young: Nutritional influences on developmental epigenetics. Canadian Federation of Biological Sciences 48th Annual Meeting, June 2005, Guelph, Ontario, Canada. Review Publications Shi, T., Wang, F., Stieren, E., Tong, Q. 2005. SIRT3, a mitochondrial sirtuin deacetylase, regulates mitochondrial function and thermogenesis in brown adipocytes. Journal of Biological Chemistry. 280(14):13560-13567. Waterland, R.A., Shi, X., Lin, J-R., Smith, C.A. 2005. Nutritional epigenetics in Axin-fused (Axin[Fu]) mice: Kinky "tales" about mom's diet [abstract]. The Federation of American Societies for Experimental Biology Conference. Part I(abstract 161.2):A218-A219. Waterland, R.A. 2005. Commentary: The global relevance of "biological Freudianism". International Journal of Epidemiology. 34(1):15-17. Harris, L.L., Bukowski, J.T., Karpen, H. 2005. Differential expression of alternate hedgehog receptor splice forms during development [abstract]. Pediatric Academic Society. 57:Abstract 1975. 2005 CDROM. Motil, K.J., Fraley, K., Schultz, R.J., Ochs, U., Sybert, V. 2005. Growth characteristics of children with ectodermal dysplasia syndromes. Pediatrics. 116(2):e229-234.